The Role of P4H in Tissue-Titanium Integration

P4H 在组织-钛整合中的作用

• 批准号: 6967136
• 负责人: TAKAHIRO OGAWA
• 金额: $ 23.18万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6967136
• 关键词: biomaterial evaluation; biomaterial interface interaction; biomechanics; bone prosthesis; bone regeneration; cell adhesion; collagen; enzyme activity; enzyme linked immunosorbent assay; gene expression; genetic transcription; hardness; laboratory rat; medical implant science; normal ossification; nuclear runoff assay; osteogenesis; oxygenases; polymerase chain reaction; protein structure; tissue /cell culture; titanium; transfection; wound healing

DESCRIPTION (provided by applicant): The structure and mechanisms of bone-implant integration, osteointegration, are poorly understood. Our long-term goal is to identify the biomechanical and molecular properties in bone-titanium integration and to develop improved implant technology for clinical applications. Using a differential display-polymerase chain reaction (DD-PCR) assay, we isolated and identified prolyl 4-hydroxylase alpha-subunit (P4Ha) as a gene transcript specifically upregulated in early stage of bone healing with titanium implants. P4H functions during post-translational hydroxylation of collagen chains, a process essential for cross-linking individual collagen molecules into a trimetric mature collagen to prevent intracellular degradation. The objective of this application is to identify the role of P4H in establishing tissue-titanium integration. We recently discovered that mineralized tissue cultured on titanium, especially acid-etched, roughened titanium, is harder than that cultured on a polystyrene surface. Our studies have also demonstrated characteristic collagen deposition dynamics on titanium. We hypothesize that early increased P4Ha gene expression in mineralized tissue on titanium is responsible for the observed collagen deposition dynamics and enhanced hardness and interfacial adhesion of the tissue on titanium. The following specific aims are formulated to test this thesis. Specific Aim 1: To establish the role of P4H on collagen deposition dynamics in the mineralized tissue. We will study loss-in-function by inhibiting P4H function with the hydroxylation inhibitor, 3,4 dehydroproline at early, mid and late stages of osteoblastic culture with and without titanium. Gain-in-function will be studied by transfecting the plasmid expression vector encoding P4Ha into osteoblasts at early and mid stages of culture. We will determine the density and localization of collagen deposition change in the various culture conditions. Changes in the expression of representative osteoblastic genes, cellular proliferation, mineralization and tissue surface morphology will also be examined. Specific Aim 2: To establish the role of P4H on the biomechanical properties of mineralized tissue and the relationship between hypoxia and P4H. We have shown that enhanced biomechanical property of mineralized tissue on titanium is associated with characteristic collagen-related structural changes. Therefore, we will control collagen synthesis using P4Ha manipulation and evaluate hardness, elastic modulus, critical load (force to delaminate tissue) and interface adhesion strength. Relationships between P4H function assessed by P4H inhibition or P4Ha gene transfection and the biomechanical properties and pattern in collagen deposition will be studied. We, further, will evaluate the effectiveness of P4H-driven biomechanical enhancement on titanium having different surface topographies. Finally, to address the regulatory function of HIF on P4H, we will establish the dissolved oxygen concentration in the culture with or without titanium, HIF expression levels, and the effect of HIF expression on P4H transcription. We seek to elucidate the mechanisms underlying tissue-titanium integration and the biomechanics of integrated mineralized tissue and anticipate that identifying a link with P4H will provide an opportunity to pursue P4Ha as a potential, novel strategy for implant therapy.

描述（由申请人提供）：对骨植入物整合的结构和机制，骨整合的理解很少。我们的长期目标是确定骨titanium整合中的生物力学和分子特性，并为临床应用开发改进的植入技术。使用差异显示聚合酶链反应（DD-PCR）测定法，我们分离并鉴定出脯氨酰4-羟化酶α-亚基（P4HA）作为一种基因转录本在用钛植入物的骨骼愈合的早期阶段中上调的基因转录本。 P4H在胶原蛋白链翻译后的羟基化过程中起作用，这是将单个胶原蛋白分子交联成至少成熟的成熟胶原蛋白以防止细胞内降解至关重要的过程。该应用的目的是确定P4H在建立组织titanium整合中的作用。我们最近发现，在钛上培养的矿化组织，尤其是酸蚀刻，粗糙的钛，比在聚苯乙烯表面培养的组织要难。我们的研究还证明了钛对钛的特征性胶原沉积动力学。我们假设早期增加了钛矿化组织中的P4HA基因表达，导致观察到的胶原沉积动力学，并增强了钛组织组织组织的硬度和界面粘附。提出以下特定目标以检验该论文。具体目的1：确定P4H在矿化组织中胶原蛋白沉积动力学中的作用。我们将通过用羟基化抑制剂抑制P4H功能，在成骨细胞培养的早期，中期和晚期抑制P4H功能，使用和不含钛。通过将编码P4HA的质粒表达载体转染到培养物的早期和中期，将研究函数。我们将确定各种培养条件下胶原沉积变化的密度和定位。还将检查代表性成骨基因，细胞增殖，矿化和组织表面形态的表达变化。具体目的2：确定P4H对矿化组织生物力学特性的作用以及缺氧与P4H之间的关系。我们已经表明，矿化组织在钛上的生物力学特性增强与特征性胶原相关的结构变化有关。因此，我们将使用P4HA操纵来控制胶原蛋白合成，并评估硬度，弹性模量，临界负载（将组织的力）和界面粘附强度。通过P4H抑制或P4HA基因转染评估的P4H功能与胶原沉积中的生物力学特性和模式之间的关系。此外，我们将评估P4H驱动的生物力学增强对具有不同表面地形不同的钛的有效性。最后，为了解决HIF对P4H的调节功能，我们将在有或没有钛，HIF表达水平以及HIF表达对P4H转录的影响的培养中建立溶解的氧浓度。我们试图阐明组织核核整合的基础机制和综合矿化组织的生物力学，并预计确定与P4H的联系将为追求P4HA作为植入疗法的潜在新型策略提供机会。